【作者】 杨和平；

【导师】 胡治平；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 研究背景阿尔茨海默病(Alzheimer’s disease,AD)的典型神经病理特征是患者脑内出现以β-淀粉样蛋白(amyloid beta peptide,Aβ)为主要成分的神经元外老年斑(senile plaque,SP)、神经元内神经原纤维缠结(neurofibrillary tangles,NFTs)、广泛的神经元变性消失。神经元变性凋亡涉及信号转导、基因调控和凋亡效应等三个阶段。信号转导过程的激活是启动细胞变性凋亡的必要前提,从而调控细胞存活与死亡。神经元变性凋亡的发生机制主要涉及两条信号转导通路,即线粒体通路和膜死亡受体通路。二者最后都启动caspase的级联反应而引起神经元凋亡。此外,细胞周期异常也可以影响到神经元的存活。神经元内外存在着在各种致凋亡因素,同时也存在着抗凋亡的相关信号途径,其中经典的抗凋亡信号途径是磷脂酰肌醇-3激酶(phosphoinositide-3kinase,P13K)/丝氨酸-苏氨酸蛋白激酶(Akt)信号通路。细胞间黏附分子5(Intercellular adhesion molecule-5,ICAM-5,telencephalin)是一种在哺育动物端脑神经元体树突膜特异性表达的复杂黏附分子,具有促进神经元树突生长与影响学习记忆功能的作用。ICAM-5的胞质区结合于埃兹蛋白-根蛋白-膜突蛋白(ezrin-radixin-moesin,ERM)家族蛋白使膜蛋白与细胞骨架肌动蛋白连接起来,而ERM的磷酸化活性形式可以启动PI3K/Akt信号通路。因此,ICAM-5可能通过激活ERM蛋白而启动PI3K/Akt信号通路。但其是否可以对抗Aβ的神经毒性或参与信号传导,目前国内外无这方面的报道。研究目的探索ICAM-5是否减轻或缓解Aβ的神经毒性作用和激活抗凋亡的PI3K/Akt信号通路来阻断Aβ诱导的凋亡信号传导,为AD和其他神经变性疾病的防治提供相关基础。研究方法1.建立神经元模型:用常表达人野生型ICAM-5蛋白的人神经母细胞瘤细胞PAJU-ICAM-5进行连续不加压传代,使其质粒脱失,经免疫荧光和Western Blot鉴定为PAJU细胞后转染含NEO抗性基因的空载体,建立空载体转染的对照细胞模型PAJU-NEO;2.用Aβ作为神经毒性刺激物,分别对PAJU-ICAM-5和PAJU-NEO细胞进行不同浓度和时间段刺激,采用MTT法检测神经元活力、Hoechst 33258神经元核染色观察核凋亡、流式细胞仪检测神经元凋亡,以了解ICAM-5是否具有保护神经元对抗Aβ的整体作用;同时用蛋白免疫印迹技术检测Aβ对ICAM-5表达的影响;3.测量在不同浓度和时间段Aβ作用下的神经突起,并使用免疫荧光染色,观察Aβ对神经突起末梢的影响,以了解CAM-5表达是否具有保护神经突起对Aβ的局部作用;4.采用免疫印迹技术检测Aβ刺激作用后磷酸化蛋白的表达,以了解抗凋亡变性信号途径是否被激活。研究结果1.PAJU-ICAM-5细胞经不加压连续传代10代后出现质粒完全脱失,经过免疫荧光检测和Western Blot验证无ICAM-5表达;空载体pcDNA3.1转染的PAJU-NEO细胞株建立。2.ICAM-5蛋白常量稳定表达于PAJU细胞后,可减轻Aβ诱导的PAJU细胞凋亡变性,表现为细胞存活率升高和凋亡率降低。同时,表达于PAJU细胞中的ICAM-5蛋白在经受长时间高浓度Aβ42作用后出现降低趋势。3.PAJU-ICAM-5细胞突起分支长度损伤较PAJU-NEO细胞明显减轻。4.通过免疫印记技术发现,ICAM-5蛋白的表达使Moesin蛋白的558位苏氨酸(Thr558)残基磷酸化而形成活化状态,经Aβ刺激后引起其家族蛋白中的Ezrin蛋白的第567位苏氨残基酸(Thr567)和Radixin蛋白的564位苏氨酸(Thr564)出现磷酸化激活,并由此导致了PI3K蛋白调节亚单位p85α的508位酪氨酸残基(Tyr508,p85α)磷酸化,引起PI3K活化,PI3K直接促使Akt蛋白的473位丝氨酸(Ser-473)残基磷酸化而激活了信号通路。5.PI3K特异性抑制剂LY294002不仅下调PI3K蛋白表达,而且还可以下调ICAM-5、磷酸化ERM、磷酸化PI3K及磷酸化Akt蛋白的表达,而总ERM、总PI3K及总Akt却表达于未经LY294002和Aβ处理以及经过LY294002和Aβ处理过的PAJU细胞。提示LY294002可能是这条信号通路上的生物学调节标志。研究结论1.我们首次证明了ICAM-5蛋白常表达具有神经保护作用,可以减轻Aβ神经毒性所致的PAJU细胞凋亡及神经突起变性。2.Aβ42与ICAM-5之间具有相互作用,长时间高剂量的Aβ42使ICAM-5表达下调。3.首次发现ICAM-5通过激活ERM而激活PI3K/Akt信号途径,因此减轻Aβ诱导的神经变性。4.LY294002可能是ERM/PI3K/Akt信号通路上的生物学调节点。更多还原

【Abstract】 Backgroud Alzheimer’s disease（AD） is a neurodegenerative disorder pathologically by accumulation of amyloid beta protein（Aβ）,a major constituent of extracellular senile plaque（SP） and intracellular neurofibrillary tangles（NFTs） and loss of cholinergic neurons.Neuronal degeneration and apoptosis are involved in three stage including signal transduction,gene regulation and apoptosis execution.Activation of signal transduction procedure is the essential condition of initiating neuron apoptosis and regulates cell survival and death.The mechanism of neuronal degeneration or apoptosis includes two major cell death signaling pathways which are mitochondrial death pathway and death receptor pathway.The two pathways start caspase cascade reaction at last and lead neurons death. Besides,abnormal of cell cycle also affects neuron survival.In the process of neurons apoptosis,there also exist relative anti-apoptosis signaling pathways.The typical one is phosphatidylinositol 3-kinase（PI3K）/Akt pathway.Intercellular adhesion molecule-5（ICAM-5,telencephalin） is a cell adhesion molecule expressed in the somatodendritic membrane of telencephalic neurons in mammalian brain.It induces dendritic outgrowth and is involved in regulation of memory formation and learning.ICAM-5 cytoplasmic region binds ERM（ezrin/radixin/moesin） family proteins that link membrane proteins to actin cytoskeleton.And phosphorylation of ERM can initiate PI3K/Akt pathway.But there is no research on whether ICAM-5 can abate Aβneurotoxicity or is involved in signaling transduction.Objective To explor whether ICAM-5 can abate neurotoxicity of Aβand initiate PI3K/Akt pathway,and provide relative basis for prevention and cure of AD and other neurodegenerative diseases.Methods 1.The control neuronal model PAJU-NEO was established by transfected PAJU cells with empty vector pcDNA3.1.And PAJU cells were obtained by successive passage of PAJU-ICAM-5 without pressure selection.It was identified by immunofluorescence and western blotting.2. We treated PAJU-ICAM-5 and PAJU-NEO cells with different concentrations of Aβin different times.To detect neuronal survival rate with MTT and apoptosis rate with Hoechst 33258 and flow cytometry. Western blotting was used to find out effect of Aβon expression of ICAM-5.3.To measuer process of PAJU-ICAM-5 and PAJU-NEO cells cultured in different concentrations of Aβin different times.And immunofluorescence was used to observe neurites damage.4.Western blotting was used to find out phosphorylation of Ezrin（Thr567）,Radixin （Thr564） and Moesin（Thr558）,PI3K（Tyr508,p85α） and Akt（Ser473）. Results 1.Neuronal model PAJU-NEO was established and express neo resistivity.2.We discovered that nomalexpression of ICAM-5 can protect neurons from Aβneurotoxicity(Aβ₄₂ 1.0nmol/L) induced apoptosis, as indicated by increase neuronal survival rate and decrease damage of neurites(Aβ₄₂ and Aβ₃₅ 0.5,0.8nmol/L) and apoptosis rate.Meanwhile, ICAM-5 expression down regulation present due to long time expose to Aβ. 3.Furthermore,ICAM-5 interacted with ezrin-radixin-moesin（ERM） protein family in PAJU cells,and active phosphorylation of Ezrin（Thr567）, Radixin（Thr564） and Moesin（Thr558）.Phosphorylated ERM activated phosphorylation of PI3K（TyrS08,p85α）,which made activation of Akt （Ser473） directly.4.Western blot analysis showed that 30μM of the LY294002 markedly inhibited ICAM-5,phospho-ERM,phospho-PI3K and phospho-Akt in PAJU cells,while the total ERM,PI3K and Akt expression in both PAJU-ICAM-5 and PAJU-NEO cell lines was identical in nontreated and treated cells.It suggested that LY294002 play a role on this pathway in the biological regulation of this marker.Conclusion 1.Here we demonstrate for the first time that nomalexpression of ICAM-5 in human neuroblastoma PAJU cells can protect the neurons from Aβneurotoxicity as indicated by reduce apoptosis and neurites degeneration,increase neuronal survival rate.2.Long time expose to Aβmay reduce expression level of ICAM-5.3.We also demonstrate for the first time that ICAM-5 attenuates amyloid beta protein induced neuronal degeneration by ERM/PI3K/Akt pathway.4.LY294002 may be a biological regulative marker of ERM/PI3K/Akt pathway.更多还原